Optimising protein detection with fixable custom oligo-labelled antibodies for single-cell multi-omics approaches

Background and Aim Single-cell RNA sequencing (scRNA-seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of oligonucleotide-labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA-seq-only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co-functioning oligonucleotide-labelled antibodies. Main Methods and Results We present here a simple adjustable oligonucleotide-antibody conjugation method which enables a desired level of oligo-conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA-seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo-antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA-seq. Conclusions and Implications Optimised oligo-labelling will improve detection of weak protein targets in scRNA-seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE-seq libraries. Furthermore, the use of a pre-stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA-seq multimodal experiments.Peer reviewe

Regulation of the complement system and immunological tolerance in pregnancy

Preeclampsia is a serious vascular complication of the human pregnancy, whose etiology is still poorly understood. In preeclampsia, exacerbated apoptosis and fragmentation of the placental tissue occurs due to developmental qualities of the placental trophoblast cells and/or mechanical and oxidative distress to the syncytiotrophoblast, which lines the placental villi. Dysregulation of the complement system is recognized as one of the mechanisms of the disease pathology. Complement has the ability to promote inflammation and facilitate phagocytosis of placenta-derived particles and apoptotic cells by macrophages. In preeclampsia, an overload of placental cell damage or dysregulated complement system may lead to insufficient clearance of apoptotic particles and placenta-derived debris. Excess placental damage may lead to sequestration of microparticles, such as placental vesicles, to capillaries in the glomeruli of the kidney and other vulnerable tissues. This phenomenon could contribute to the manifestations of typical diagnostic symptoms of preeclampsia: proteinuria and new-onset hypertension. In this review we propose that the complement system may serve as a regulator of the complex tolerance and clearance processes that are fundamental in healthy pregnancy. It is therefore recommended that further research be conducted to elucidate the interactions between components of the complement system and immune responses in the context of complicated and healthy pregnancy.Peer reviewe

Different selectivities of oxidants during oxidation of methionine residues in the α-1-proteinase inhibitor

AbstractOxidation of the reactive site methionine (Met) in α-1-proteinase inhibitor (α-1-PI) to methionine sulfoxide (Met(O)) is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in α-1-PI, we measured the molar ratio Met(O)/α-1-PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H2O2/chloride system and the related compound NH2Cl. With taurine monochloramine, another myeloperoxidase-related oxidant, 1.05 mol Met(O) were generated per mol α-1-PI during inactivation. These oxidants attack preferentially one Met residue in α-1-PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase-derived oxidants. In contrast, the ratio found for ozone and m-chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the reactive site Met in α-1-PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of α-1-PI. Ozone and m-chloroperoxybenzoic acid were 10-fold less effective and the superoxide anion/hydroxyl radicals were 30–50-fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase-derived oxidants. The myeloperoxidase-related oxidants are discussed as important regulators of α-1-PI activity in vivo

Properdin binds independent of complement activation in an in vivo model of anti-glomerular basement membrane disease

Properdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent in vitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin in vivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3-and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues in vivo independent of its main ligand C3.Peer reviewe

Antibody responses to immunoevasion proteins BBK32 and OspE constitute part of the serological footprint in neuroborreliosis but are insufficient to prevent the disease

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is the most common tickborne disease. Its neuronal form, neuroborreliosis, comprises 3 to 38% of borreliosis cases in Europe. Borrelia outer surface proteins and virulence factors, OspE and BBK32, have been previously reported to help cause infection by promoting attachment to human host epithelial cells and evading complement attack. We assessed the serological responses to BBK32 and OspE in 19 individuals diagnosed with neuroborreliosis to see whether antibodies that could both target the bacteria and neutralize the virulence mechanisms on the microbial surface emerge. Results evaluate levels of total protein, IgG and the chemokine CXCL13, a determinant for B-cell recruitment during neuroinflammation, in patients' cerebrospinal fluid samples. Antibody levels against BBK32 and OspE correlated with those against VlsE, a well-characterized diagnostic serological marker of the disease. A dual serological profile of the patients was observed. K-means clustering split the cohort into two discrete groups presenting distinct serological and CNS responses. One group contained young patients with low levels of anti-BBK32 and OspE antibodies. The other group showed stronger responses, possibly following prolonged infections or reinfections. Additionally, we assessed anti-ganglioside antibodies that could cause autoimmunity or complement dysregulation but observed that they did not correlate with neuroborreliosis in our patient cohort. The dual nature of antibody responses against the virulence factors BBK32 and OspE in neuroborreliosis patients may suggest the necessity of repeated exposures for efficient immune responses. Better protection could be achieved if the virulence factors were formulated into vaccines.</p